This grant proposal requests a fourth cycle of funding to explore and expand our understanding of the genetic basis of Hirschsprung disease (HSCR). From the genesis of these studies until today, we have established HSCR as a model complex disorder by: (1) demonstrating that HSCR shows oligogenic inheritance; (2) discovering at least 6 (RET, GDNF, EDNRB, EDN3, SOX10, SEMA3) of the 12 genes known for this disorder; (3) demonstrating genetic interactions between RET and EDNRB signaling (in humans and mouse models) and their downstream transcriptional consequences, as an explanation of the reduced penetrance; (4) discovering the role of common polymorphisms at RET and SEMA3 on HSCR and the molecular basis of this association by in vitro and in vivo analyses; and, (5) assembling and maintaining a database/sample repository (International Hirschsprung Disease Consortium) and a public information resource for ascertaining new cases and families. This grant describes four sets of specific aims that have the goal of identifying a large number of susceptibility genes for HSCR by virtue of them harboring many more dosage and sequence mutations than would be expected by chance. First, we will confirm our recent discovery of SEMA3 as a HSCR locus; second, we will perform focused genetic screens of dosage mutations at all human genes and a genome-wide association study for common susceptibility variants; third, we will target DNA sequencing on a set of 2,000 genes and target loci (~10% of the coding genome) that have emerged from these prior genetic screens. A fourth aim will be to continue sampling new cases and families, over and above those being collected by members of the International Hirschsprung Disease Consortium, and expand our information resource. We have focused this research on gene discovery and on primary functional analysis leading to specific identification of genes, rather than a deeper understanding of mechanisms of pathogenesis. This choice, a minor departure from our tactic in the last cycle and not possible before, is occasioned by the realization that we have identified very few of the HSCR genes and that many more can now be discovered using new technologies. A better map of this 'HSCR gene universe' will allow a better choice of which mechanisms to investigate in a deeper fashion. PUBLIC HEALTH RELEVANCE: Hirschsprung disease (HSCR) is the most common birth defect due to a functional intestinal obstruction. Molecular genetic studies are ideal for identifying the cellular defects in enteric neuroblasts and their interacting cells intrinsic to this disorder. We propose major innovative molecular studies for identifying the specific genes and how they lead to HSCR.